This project will continue to focus on the identification of environmental and genetic risk factors for development of hepatocellular carcinoma (HCC) in Taiwan. We are carrying out a case-control study nested in a cohort established by our collaborator, Dr. Chien in which 25,611 subjects were recruited between 1990 and 1992. In our ongoing studies, we demonstrated that, in conjunction with hepatitis B or C virus infection, aflatoxin B1 (AFB1), 4-aminobiphenyl, and polycyclic aromatic hydrocarbons (PAH) increased HCC risk. Synergistic interactions between virus and chemical were also observed. Genotyping for polymorphisms in carcinogen metabolism, oxidative stress and DNA repair are ongoing and suggest that genetic susceptibility is also significant. We will continue to analyze blood and urine samples for AFB1, and PAH biomarkers in all new cases and matched controls but also expand to biomarkers of oxidative stress including oxidized plasma proteins and urinary 8-oxodeoxyguanosine and isoprostane. We hypothesize that biomarkers of environmental exposure and oxidative stress will be higher in cases than controls. We will also continue to genotype subjects for polymorphisms in the pathways currently being investigated but expanding the number of genes. We hypothesize that cases will more frequently be carriers of "higher risk" alleles than controls and that there will also be an interaction between environmental exposures and genotype. Finally, our pilot study demonstrated that p16 was frequently methylated in tumor DNA isolated from plasma of HCC cases. We will now carry out a case-control study within the cohort to determine the frequency of methylation of a panel of genes. We hypothesize that methylation will be more frequent in cases than controls. These studies will allow the development of a panel of genes in which the detection of methylation in blood DNA identifies HCC earlier than currently possible. Our collaborative study with Dr. Chen has provided much useful data on environmental and genetic risk factors for cancer development.